We thank all members of UMR 177 INTERTRYP IRD CIRAD Universit&#233; de Bordeaux. This research was supported by internal funding from University of Bordeaux and support from the ANR, LABEX ParaFrap ANR-11-LABX-0024, and from the Association pour le d&#233;veloppement de la recherche en parasitologie et m&#233;decine tropicale and the Service de coop&#233;ration et d&#39;action culturelle de l&#39;Ambassade de France &#224; Bangui (Centrafrique).

Investigations conformed to the Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85&#177;23, revised 1996). Protocols were approved by our local ethics committee.
1. Animals
<ol>
	<li>Keep female Swiss (OF-1) mice aged eight to ten weeks old, 20-25 g, in an animal housing facility fifteen days before each experiment. House them in ventilated boxes that are kept in a protected, temperature (22 &#176;C) and humidity (50%) controlled room, with 12 hours on/off light cycle.</li>
	<li>Give animals free access to food and water. Minimize pain, suffering and distress and provide enrichment of the environment.</li>
	<li>For housing, use clear-walled cages, enrichment with wooden sticks and cardboard tunnels. Gently draw an animal into a tunnel to transfer it from the cage to the palm of the hand.</li>
	<li>Perform daily monitoring to assess signs of prostration, social isolation, body injury, ruffled hair, or lack of grooming.</li>
	<li>Weigh each animal once per week. Perform regular inspections by a veterinarian.</li>
	<li>For natural parasites, collect blood at the peak of parasitemia and for parasites causing animal death, collect blood the day before presumed death. 
	NOTE: All experiments with infectious agents are performed in dedicated rooms, according to university approved guidelines.</li>
</ol>
2. Buffers, media preparations
<ol>
	<li>Weigh out each substance and add distilled water for the following buffers:
	<ol>
		<li>Prepare Concentrated Phosphate-Buffered Saline (2x): 
		Na2HPO4 (anhydrous) (MW 141.96 g) 10.14 g 
		NaH2PO4&#8729;2H2O (MW 156.01 g) 0.62 g 
		NaCl (MW 58.44 g) 2.55 g 
		Distilled H2O to 1 L</li>
		<li>Prepare Phosphate-Buffered Saline-Glucose: 
		Na2HPO4 (anhydrous) (MW 141.96 g) 5.39 g 
		NaH2PO4&#8729;2H2O (MW 156.01 g) 0.31 g 
		NaCl (MW 58.44 g) 1.70 g 
		Glucose (MW 180 g) 10 g 
		Distilled H2O to 1 L</li>
		<li>Prepare 1 M KH2PO4.</li>
		<li>Prepare Elution Buffer: Supplement Phosphate-Buffered Saline-Glucose with 
		Penicillin (100 U/mL), Streptomycin (100 &#181;g/mL), and Phenol red (5 &#181;g/mL).</li>
	</ol>
	</li>
</ol>
3. Preparation of DEAE-cellulose
<ol>
	<li>Plan around 5 hours for the DEAE-cellulose preparation.</li>
	<li>Wash 100 g of DEAE-cellulose with distilled water in a flask with a narrow neck and allow to settle, then discard fine particles. Repeat washes until the supernatant is clear.</li>
	<li>Add 3 L of concentrated 2x Phosphate-Buffered Saline and stir.</li>
	<li>Adjust pH to 8.0 with 1 M KH2PO4 and discard supernatant.</li>
	<li>Wash twice with distilled water and leave to settle.</li>
	<li>Wash and allow to settle twice with 3 liters Phosphate-Buffered Saline-Glucose and discard the supernatants.</li>
	<li>Measure the volume of cellulose and add an equal volume of Phosphate-Buffered Saline-Glucose, distribute into plastic bottles and store at -20 &#176;C.</li>
</ol>
4. Parasites
<ol>
	<li>Collect trypanosome strains in areas endemic for human and animal trypanosomiasis. Keep parasites frozen in liquid nitrogen. 
	NOTE: Trypanosoma musculi is non-pathogenic to humans and is an extracellular trypanosome used to safely replace pathogenic trypanosomes in various laboratory experiments.</li>
	<li>In the case of laboratory investigations requiring human pathogens, handle experiments with care in dedicated appropriate biohazard safety level (BSL) conditions and precautions;&#160;BSL2 for T. b. gambiense and BSL3 for T. b. rhodesiense. In field conditions, standard microbiological practice is established: secure sampling, mechanical pipetting, frequent decontamination of surfaces, and sterilization of waste.</li>
</ol>
5. Mouse infection
<ol>
	<li>Rapidly thaw parasites in a water bath at 37 &#176;C.</li>
	<li>Observe a drop of thawed infected blood with a microscope. Assess parasite viability by measuring the percentage of motile forms17.</li>
	<li>Inject parasites intraperitoneally into mice (0.5 mL per mouse). Each day, post-infection, collect 20 &#181;L of tail blood by needle puncture and observe under a microscope. Evaluate parasitemia according to Herbert and Lumsden18 by counting parasites in several microscope fields, or by using a&#160;haemocytometer.</li>
	<li>When the parasitemia reaches a threshold that is defined for each strain, collect the blood (1 mL/mouse) in the elution buffer (5 mL/mouse) containing heparin (10 U/mL). 
	NOTE: The original and novel work of Lanham and Godfrey reported the optimal ionic strength of phosphate buffered saline glucose for several host/parasite species from a pH 8.0 stock solution.</li>
</ol>
6. Parasite separation
NOTE: All experiments from this point onwards must be done in a tissue culture hood wearing gloves. The room temperature and humidity in the laboratories used were 22 &#176;C and 45% respectively. In field conditions, parasite separation has been successfully performed at 34 &#176;C.
<ol>
	<li>Place a 10 mL syringe in a vertical support and add a previously cut circular, piece of filter paper, glass wool or cellulose sponge.</li>
	<li>Pour the DEAE cellulose into the syringe until the 8 mL level is reached, and then wash with 25 mL of elution buffer.</li>
	<li>Carefully add 2 mL of diluted blood at the top of the column and then add elution medium. Regularly add elution buffer according to the transit of the trypanosomes.</li>
	<li>Collect effluent drops from the columns in a centrifuge tube and check regularly for the presence of parasites with a microscope.</li>
	<li>When parasites are no longer detected in the column effluent, centrifuge the tube (1,800 x g, 10 minutes, at 4 &#176;C in the laboratory and at ambient temperature in field conditions).</li>
	<li>Remove the supernatant and suspend the parasites in 1 mL of the relevant medium required for the next investigative step.</li>
	<li>Count parasites with a hemocytometer and dilute them in appropriate medium if necessary.</li>
</ol>

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The authors have nothing to disclose.

Purified trypanosomes represent a powerful means to study immunology, biochemistry, cellular and molecular biology. Large expanses of data and results have been obtained from trypanosomes, which has then helped to obtain information from other eukaryotic cells30. Trypanosomes are also the subject of important and interesting research because they have devised numerous mechanisms that permit them to survive and grow in two very different environments: the tsetse fly vector and the mammalian host<su...

The method presented described here allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits robust serological and research investigation.
HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense1. These protozoan parasites multiply extracellularly in the bloodstream, lymph, and interstitial fluids during the first stage of the disease (hemolymphatic stage). The second stage (meningoencephalitic stage) begins when parasites cross the blood brain barrier; neurological signs, including a sleep disorder, which has given its name &#34;sleeping sickness&#34; to this disease, are typical of this second-stage2. Related trypanosomes (T. evansi, T. congolense, T. vivax, T. b. brucei) are the causative agents of animal African trypanosomosis (AAT)3.
The World Health Organization (WHO) aims to eliminate HAT as a public health problem by 2020 and to stop transmission by 20304. The recent introduction of rapid diagnosis tests has improved serological diagnosis1,4,5. Several molecular diagnostic tests have been developed but their role in field diagnostics has not yet been established5. They are used to identify the sub-species of the brucei group and atypical trypanosomiasis caused by parasites responsible for animal trypanosomosis6.
The detection of the parasite is essential for the diagnosis, treatment and follow-up, as serology can give false positive and unfortunately false negative results1. The direct microscopical observation of these hemoflagellate protists is difficult in HAT cases that are caused by T. b. gambiense, (more than 95% of cases) as low parasitemias are the rule, whereas for HAT caused by T. b. rhodesiense, a large number of parasites are frequently present in the blood. Various concentration techniques have been used, such as thick drop and capillary tube centrifugation (CTC), but the separation of parasites from blood by a column of anion-exchanger (DEAE cellulose) followed by centrifugation and microscopic observation of the pellet, is the most sensitive method (around 50 parasites/mL of blood can be detected)1,7. Consequently, the purification of trypanosomes by this anion-exchangers (DEAE cellulose) method is the best and, to date, the reference method for visualizing and isolating parasites from blood for HAT diagnosis. In field conditions, a mini-column of DEAE cellulose has been successfully used and several improvements have facilitated microscopical observation7,8.
The method of trypanosome separation from blood, described below, depends on parasite surface charge, which is less negative than mammalian blood cells9. Interestingly, this method was developed 50 years ago, in 1968 by Dr. Sheila Lanham, and remains the gold standard for detection and preparation of bloodstream trypanosomes. It is fast and reproducible for salivarian trypanosomes from a wide range of mammals, permitting the diagnosis of both animal and human trypanosomiasis10.
To obtain living, purified parasites, infected blood is added onto an anion-exchanger column. Chromatography conditions (mainly pH, ionic strength of buffers/media) have to be adapted to each trypanosome species, and more generally, to each mix of mammalian blood cells and trypanosomes10. Elution buffer is precisely adjusted to pH 8 for most African trypanosomes10. This method favors the concentration of parasites found in the blood of patients, because parasitemias can be too low to be detected by microscopic observation alone, and it also enables laboratory investigations. Working with freshly isolated trypanosomes and on blood from infected animals, using this technique, is more pertinent for various investigations than studies with parasites that have been cultured in axenic conditions in the laboratory for an indefinite period.
Host-parasite relationships are best studied with a parasite infecting its natural host, therefore, T. musculi, a natural murine parasite, which is representative of extracellular trypanosomes, has many advantages as murine infection involves in a small laboratory animal and does not require biohazard safety level (BSL) conditions. T. musculi does not kill immunocompetent mice, unlike many other Trypanosoma species, including human pathogens. T. musculi are not eliminated in T cell-deprived mice and parasitemias can be increased&#160;in infected mice by modifying food and nutrient intake11. This parasite modulates the immune response in co-infections with other pathogens12. T. musculi from infected mice exhibit differences from cultured T. musculi, for example, the expression of membrane Fc receptors is lost in T. musculi axenic cultures, compared to parasites purified from infected mice13,14. Excreted-secreted factors (ESF) are also qualitatively and quantitatively less expressed in axenic trypanosome cultures and differ between strains isolated in endemic areas15. ESF are the first antigens to be displayed to the host immune system and so play an important role in the initial host immune response16.
In experimentally infected animals for laboratory investigations, this protocol facilitates experimentation on a greater number of parasites, minimizing the number of mice required especially when using immunosuppressed animals. The variant surface glycoproteins (VSGs) that are used in the Card Agglutination Test for Trypanosomiasis (CATT) in mass screening are still purified from trypanosomes that are propagated in rats. The two rapid diagnostic tests (individually wrapped cassettes) that are now available for use in the field, are still using an infective model source of native VSGs and not in vitro cultured trypanosomes1,4,5. The advancement in the study of trypanosome immunology and biology has been facilitated since these DEAE cellulose purified parasites can be easily obtained in large quantities from naturally or experimentally infected hosts, and in particular, rodents.

<table><tbody><tr><td>10 mL Pipettes&amp;nbsp;</td><td>Falcon</td><td>357,551</td><td></td></tr><tr><td>2 mL Pipettes&amp;nbsp;</td><td>Falcon</td><td>352,507</td><td></td></tr><tr><td>Centrifugation tube 50 mL</td><td>Falcon</td><td>352,070</td><td></td></tr><tr><td>Centrifuge</td><td>Sigma Aldrich</td><td>4K15</td><td></td></tr><tr><td>DEAE cellulose</td><td>Santa Cruz</td><td>s/c- 211213</td><td>100 G</td></tr><tr><td>filter paper&amp;nbsp;</td><td>Whatman</td><td>1,001,125</td><td></td></tr><tr><td>Flat bottom flask narrow neck</td><td>Duran</td><td>21 711 76</td><td>6000 mL</td></tr><tr><td>Glucose&amp;nbsp;</td><td>VWR</td><td>101174Y</td><td>500 G</td></tr><tr><td>Heparin</td><td>Sigma Aldrich</td><td>H3149-50KU</td><td>5 000 U</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>VWR</td><td>120 26936.260</td><td>500 G</td></tr><tr><td>Microscope</td><td>Olympus</td><td>CH-20</td><td></td></tr><tr><td>Microscope coverslips</td><td>Thermofisher scientific</td><td>CB00100RA020MNT0</td><td></td></tr><tr><td>Microscope slides</td><td>Thermofisher scientific</td><td>AGAA000001</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>VWR</td><td>100 28026;260</td><td>500 G</td></tr><tr><td>NaCl</td><td>VWR</td><td>27800.291</td><td>1 KG</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>VWR</td><td>110 33616;262</td><td>500 G</td></tr><tr><td>Nalgene Plastic Media Bottles size 125 mL</td><td>Thermofisher scientific</td><td>342024-0125</td><td></td></tr><tr><td>Nalgene Plastic Media Bottles size 500 mL</td><td>Thermofisher scientific</td><td>342024-0500</td><td></td></tr><tr><td>Pasteur Pipette</td><td>VWR</td><td>BRND125400</td><td></td></tr><tr><td>Penicillin 10,000 UI/Streptomycin 10,000 &amp;micro;g</td><td>EUROBIO</td><td>CABPES01 OU</td><td>100 mL</td></tr><tr><td>Phenol red</td><td>Sigma Aldrich</td><td>P0290</td><td>100 mL</td></tr><tr><td>Syringue&amp;nbsp;</td><td>Dutscher</td><td>SS+10S21381</td><td></td></tr><tr><td>Tissue culture hood</td><td>Thermoelectro Corporation</td><td>MSC-12</td><td></td></tr><tr><td>&lt;em&gt;Trypanosoma brucei brucei&lt;/em&gt;</td><td>Institute of Tropical Medicine (Antwerp, Belgium).</td><td></td><td>ANTAT 1.1</td></tr><tr><td>&lt;em&gt;Trypanosoma brucei gambiense&lt;/em&gt;</td><td>Institute of Tropical Medicine (Antwerp, Belgium).</td><td></td><td>ITMAP 1893</td></tr><tr><td>&lt;em&gt;Trypanosoma musculi&lt;/em&gt;</td><td>London School of Hygiene and Tropical Medicine (UK)</td><td></td><td>Partinico II</td></tr></tbody></table>

purification of extracellular trypanosomes, including african, from blood by anion-exchangers (diethylaminoethyl-cellulose columns)

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations.
HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity.
Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal.
These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

Purified trypanosomes have been used in pharmaceutical tests. Parasites are transferred into culture wells containing serial dilutions of specific drugs, either alone or mixed19. Microscopic observations, evaluating motility is a marker of viability, can be performed when only a few dugs are being tested, whereas AlamarBlue cell viability assay is an excellent method for large motility assays during drug screening20. The effect&#160;of penta...

Watch this Scientific Journal Video about Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns at JoVE.com

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns

This method of trypanosome separation from blood depends on their surface charge being less negative than mammalian blood cells. Infected blood is placed and treated on an anion-exchanger column. This method, the most fitting diagnostic for African trypanosomiasis, provides purified parasites for immunological, biological, biochemical, pharmaceutical and molecular biology investigations.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is ...

purification-extracellular-trypanosomes-including-african-from-blood

Research

JoVE Journal

Immunology and Infection

10.1K Views.  Univ. Bordeaux UMR INTERTRYP 177 IRD CIRAD. Afrikaanse trypanosomen zijn verantwoordelijk voor menselijke Afrikaanse trypanosomiasis, of slaapziekte en dierlijke Afrikaanse trypanosomiasis. De trypanosomen die slaapziekte veroorzaken zijn tseetsevlieg overgedragen protist parasieten aanwezig in veel geografische foci in Afrika bezuiden de Sahara. De detectie van de parasiet is essentieel voor de diagnose, behandeling en follow-up.Een serologie kan geven vals positief en helaas, valse negatieve resultaten. Om detectie in het bloe...

Video: Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme&#39;s protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F1-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F1-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F1-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F1-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F1-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F1-ATPase isolation from other organisms.

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

This protocol describes the purification of F1-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic studies.

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to ...

Biochemistry

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Biology

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous aspiration of cells and/or microorganisms from palpable masses, organs or effusions (fluid accumulation in a body cavity) using a thin needle similar to the regular needle used for the venous puncture. The material collected by FNA is in general highly cellular, and the retrieved aspirate is then smeared, air dried, wet-fixed, stained and observed under a microscope. In the clinical context, FNA is an important diagnostic tool that serves as a guide to the appropriate therapeutic management. Because it is simple, fast, minimally invasive and requires limited investment in the laboratory and human resources, it is extensively used by veterinary practitioners, mostly in domestic, but also in farm animals. In studies using animal models, FNA has the advantage that it can be performed repeatedly in the same animal, enabling longitudinal studies through the collection of cells from tumors and organs/tissues over the course of the disease. In addition to routine microscopy, retrieved material can also be used for immunocytochemistry, electron microscopy, biochemical analysis, flow cytometry, molecular biology or in vitro assays. FNA has been used to identify the protozoan parasite Trypanosoma brucei in the gonads of infected mice, opening the possibility for a future diagnosis in cattle.

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration is a technique, whereby cells are obtained from a lesion or organ using a thin needle. Aspirated material is smeared, stained and examined under a microscope for diagnosis or used for molecular biology, cytometry or in vitro analysis. It is cheap, simple, quick and causes minimal trauma.

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation

In this protocol, we describe a method to purify human calcium-binding protein S100A12 and its ion-induced oligomers from Escherichia coli culture for immune cell stimulations. This protocol is based on a two-step chromatography strategy, which comprises protein pre-purification on an anion-exchange chromatography column and a subsequent polishing step on a hydrophobic-interaction column. This strategy produces S100A12 protein of high purity and yield at manageable costs. For functional assays on immune cells eventual remnant endotoxin contamination requires careful monitoring and further cleaning steps to obtain endotoxin-free protein. The majority of endotoxin contaminations can be excluded by anion-exchange chromatography. To deplete residual contaminations, this protocol describes a removal step with centrifugal filters. Depending on the available ion-strength S100A12 can arrange into different homomultimers. To investigate the relationship between structure and function, this protocol further describes ion-treatment of S100A12 protein followed by chemical crosslinking to stabilize S100A12 oligomers and their subsequent separation by size-exclusion chromatography. Finally, we describe a cell-based assay that confirms the biological activity of the purified protein and confirms LPS-free preparation.

This protocol describes a purification method for recombinant tag-free calcium binding protein S100A12 and its ion-induced oligomers for human monocyte stimulation assays.

In this protocol, we describe a method to purify human calcium-binding protein S100A12 and its ion-induced oligomers from Escherichia coli culture for ...

AI Program for Trypanosomiasis Detection and Classification

Superior Auto-Identification of Trypanosome Parasites by Using a Hybrid Deep-Learning Model

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of hotspot areas under active surveillance is a fundamental procedure for controlling disease transmission. Microscopic examination is a commonly used diagnostic method. It is, nevertheless, primarily reliant on skilled and experienced personnel. To address this issue, an artificial intelligence (AI) program was introduced that makes use of a hybrid deep learning technique of object identification and object classification neural network backbones on the in-house low-code AI platform (CiRA CORE). The program can identify and classify the protozoan trypanosome species, namely Trypanosoma cruzi, T. brucei, and T. evansi, from oil-immersion microscopic images. The AI program utilizes pattern recognition to observe and analyze multiple protozoa within a single blood sample and highlights the nucleus and kinetoplast of each parasite as specific characteristic features using an attention map.
To assess the AI program&#39;s performance, two unique modules are created that provide a variety of statistical measures such as accuracy, recall, specificity, precision, F1 score, misclassification rate, receiver operating characteristics (ROC) curves, and precision versus recall (PR) curves. The assessment findings show that the AI algorithm is effective at identifying and categorizing parasites. By delivering a speedy, automated, and accurate screening tool, this technology has the potential to transform disease surveillance and control. It could also assist local officials in making more informed decisions on disease transmission-blocking strategies.

Author Spotlight: AI-Driven Trypanosome Species Detection from Microscopic Images 

Worldwide medical blood parasites were automatically screened using simple steps on a low-code AI platform. The prospective diagnosis of blood films was improved by using an object detection and classification method in a hybrid deep learning model. The collaboration of active monitoring and well-trained models helps to identify hotspots of trypanosome transmission.

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of ...

Bioengineering

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

Anion-Exchange Chromatography-Based Protein Purification: A Separation Technique to Isolate a Protein of Interest From Dialyzed Bacterial Lysate Based on Net Charge

Source: Fuehner, S., et al. <a href="https://www.jove.com/v/60065">Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation</a> J. Vis. Exp. (2019)
In this video, we demonstrate an anion-exchange chromatography technique to purify human calcium-binding protein S100A12 based on the net surface charge of the protein from an Escherichia coli culture.

Source: Fuehner, S., et al. Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation J. Vis. Exp. (2019)
In this video, we ...

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

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